One of the central challenges of chemical biology remains the ability to perturb the function of any intracellular protein using a small molecule. While significant strides have been made towards developing individual ligands to specific proteins, only approximately 300 molecular targets for approved drugs have been characterized1. Furthermore, the fraction of the proteome classified as “undruggable” by current methods is estimated to be about 80%2. It is likely that many appealing drug candidates have yet to be found and that future advances in drug development will be able to overcome the boundaries of what is thought to be an “undruggable” target3,4. Therefore, the challenge for biologists remains to identify those disease-causing drug targets. To this end, advances in deep sequencing, microarray technology and genome-wide RNAi screens have been employed successfully to identify promising new drug targets. For instance, genome-wide RNAi screens have been employed to identify synthetic lethal interactions with mutated oncogenes and to identify genes necessary for various pathogenic infections5-7.
While target identification is an obvious important first step in drug development, the in vivo validation of these potential targets remains a challenge. This is due in part to the unpredictable pharmacokinetics/pharmacodynamics of any inhibitory compound identified based on in vitro inhibition of protein function. In other words, is the failure of a small molecule inhibitor to give the desired in vivo result an unforeseen consequence of its in vivo metabolism or is its target protein simply a poor drug target? To address this question, general methods are needed to functionally validate whether modulation of a putative disease-relevant protein leads to the desired in vivo result. RNAi offered initial promise for organismal validation of putative drug targets, however, the delivery and stability of duplex RNA remain major hurdles in knocking down mRNA expression in a whole animal setting8. In the absence of a direct ligand for the target protein, there are currently three categories of small molecule-based methods to control the function of a protein of interest (POI)9. First, the plant hormone auxin can be employed to dimerize a plant E3 ubiquitin ligase (TIR1) with a domain from the AUX/IAA transcriptional repressor (Aid1), which when fused to a POI can be ubiquitinated by proximity to TIR110. This method requires fusing the POI to Aid1, along with an introduction of the plant E3 ligase TIR1 into cells. A second general method used to deregulate protein function involves dimerization of FKBP12 and the FKBP12-rapamycin binding (FRB) domain from mTOR. It has been shown that a POI can be recruited to the proteasome or to the mitochondrial outer membrane by this method11-13. Again, at least two fusion proteins must be introduced into the cell for this system to function9. Lastly, two destabilizing domains (DDs), one based on the FKBP12 protein and the other on E. coli DHFR protein14,15, have been developed to destabilize a DD-POI fusion protein. The degradation conferring DD can be stabilized by inclusion of derivatives of FK50616 (in the case of mutagenized FKBP12) or the E. coli DHFR inhibitor trimethoprim (in the case of DHFR), ultimately leading to increased levels of the fusion protein. While the DD method has been successfully used in several studies17-20, it requires the continued presence of the ligand for stable expression of the fusion protein. This requirement can be a concern when studying developing embryos, which might not receive sufficient stabilizing ligand, or when studying the long term effects of a POI, in which case the ligand would have to be injected into an animal for the duration of the study. Also, in the case of the long-term expression of the POI, one must bear in mind the possible fluctuations of the POI levels that are due to the intermittent injections of the stabilizing ligand.